New ligand bearing preorganized binding side-arms interacting with ammonium cations: Synthesis, conformational studies and crystal structure

Article abstract of DOI:10.1039/b306778e

The synthesis and characterization of the new tetraazamacrocycle 4-(N),10-(N)-bis[2-(3-hydroxy-2-oxo-2H-pyridin-1-yl)acetamido]-1,7-dimethyl-1,4, 7,10-tetraazacyclododecane (L) is reported. L shows two 3-(hydroxy)-1- (carbonylmethylen)-2(1H)-pyridinone moieties as side-arms of a tetra-aza-macrocyclic base. The key coupling of side-arms was studied and the most significant results were obtained by activating the 3-(benzyloxy)-1- (carboxymethyl)-2(1H)-pyridinone as pentafluorophenol ester. The acid-base properties of L and its capability to interact with simple ammonium cations were investigated by potentiometric measurements in aqueous solution (298.1 ± 0.1 K, I = 0.15 mol dm^-3). Protonated species of L can bind NH₄⁺ or primary ammonium cations such as MeNH ₃⁺ discriminating them from secondary or tertiary ammonium cations such as Me₂NH₂⁺ or Me ₃NH⁺ which are not bound in aqueous solution. ¹H and ¹³C NMR spectra showed the existence in solution of two conformers on the NMR time scale due to the rotational restriction of the two N-C=O groups. The activation parameters were determined by dynamic variable-temperature NMR analysis. Molecular dynamics calculations gave results in agreement with the experimental data for both conformation and ammonium-binding studies, underlining that the transformation of the two secondary amines of the macrocyclic base to amide functions, forces the side-arms to remain fixed in position, almost face to face and thus to be preorganized to interact with other species. The crystal structure of the [HL]Cl·8H₂O species shows the high number of preorganized hydrogen bond sites capable, in this case, of interacting directly with five H₂O molecules.

Full text of DOI:10.1039/b306778e

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New ligand bearing preorganized binding side-arms interacting with
ammonium cations: Synthesis, conformational studies and crystal
structureyz
Mauro Formica,a Vieri Fusi,*a Luca Giorgi,a Annalisa Guerri,b Simone Lucarini,c
Mauro Micheloni,*a Paola Paoli,b Roberto Pontellini,a Patrizia Rossi,b Giorgio Tarziac
and Giovanni Zappia*c
a
Institute of Chemical Sciences, University of Urbino, Piazza Rinascimento 6, I-61029,
Urbino, Italy
Department of Energy Engineering ‘Sergio Stecco’, University of Florence, Via S. Marta 3,
I-50139, Florence, Italy
Institute of Pharmaceutical Chemistry, University of Urbino, Piazza Rinascimento 6, I-61029,
Urbino, Italy. E-mail: vieri@chim.uniurb.it
b
c
Received (in London, UK) 13th June 2003, Accepted 12th August 2003
First published as an Advance Article on the web 3rd September 2003
The synthesis and characterization of the new tetraazamacrocycle 4-(N),10-(N)-bis[2-(3-hydroxy-2-oxo-2H-
pyridin-1-yl)acetamido]-1,7-dimethyl-1,4,7,10-tetraazacyclododecane (L) is reported. L shows two 3-(hydroxy)-
1-(carbonylmethylen)-2(1H)-pyridinone moieties as side-arms of a tetra-aza-macrocyclic base. The key
coupling of side-arms was studied and the most significant results were obtained by activating the
3-(benzyloxy)-1-(carboxymethyl)-2(1H)-pyridinone as pentafluorophenol ester. The acid–base properties of L
and its capability to interact with simple ammonium cations were investigated by potentiometric measurements
+
in aqueous solution (298.1 ꢀ 0.1 K, I ¼ 0.15 mol dmꢁ3). Protonated species of L can bind NH4 or primary
ammonium cations such as MeNH3+ discriminating them from secondary or tertiary ammonium cations such
as Me2NH2 or Me3NH+ which are not bound in aqueous solution.
+
1H and 13C NMR spectra showed the existence in solution of two conformers on the NMR time scale due to
=
the rotational restriction of the two N–C O groups. The activation parameters were determined by dynamic
variable-temperature NMR analysis.
Molecular dynamics calculations gave results in agreement with the experimental data for both conformation
and ammonium-binding studies, underlining that the transformation of the two secondary amines of the
macrocyclic base to amide functions, forces the side-arms to remain fixed in position, almost face to face and
thus to be preorganized to interact with other species. The crystal structure of the [HL]Clꢂ8H2O species shows
the high number of preorganized hydrogen bond sites capable, in this case, of interacting directly with five H2O
molecules.
macrocyclic skeleton, thus forming new binding sites inside
the molecule having different hard–soft capabilities.12
Introduction
Macrocyclic compounds are continuing to attract the interest
of researchers in coordination chemistry. Indeed, the countless
possible applications of these molecules, as well as their useful-
ness in fundamental studies, have made this a thriving field of
study.1–5 These ligands are used as selective binders of metal
ions, simple models for biologically relevant systems, hosts
for guests of various nature, catalysts, NMR contrast agents,
photochemical devices, selective extractants, analytical
reagents, etc.6–8 A synthetic strategy to widen the potential
applications of macrocycles is to use them as a base on which
to attach suitable side-arms. In this way it is possible to com-
bine different chemical properties, exploiting the characteristics
of the molecular fragments connected as side-arms.9–11 The
attached moieties can also be driven to cooperate by the
3-Hydroxy-2(1H)-pyridinone and its derivative molecules
are chelating agents that have shown particular binding and
sequestering properties towards highly charged metal
cations.13 For example, they can produce decorporation of
heavy metals in animals,14 showing high affinity towards
iron(III).15 Moreover, this group shows several atoms able to
form hydrogen bonds with other species.16 This capability can
be pH-modulated in solution, producing donors or acceptors
of hydrogen bond sites.15c These moieties have usually been
attached to linear scaffolds in the 4-position by an amide
group; in the present study we used the 1,7-dimethyl-
1,4,7,10-tetraazacyclododecane as macrocyclic base upon
which we attached two 3-hydroxy-2(1H)-pyridinone molecules
linked in the 1-position to the macrocycle with a methylene-
carbonyl chain, obtaining the ligand L (Chart 1). The synthetic
aspects were investigated and new ways of attaching the side-
arm explored. In the final ligand L, the two secondary amines
of the macrocyclic base were transformed into amide groups.
In this way, these two functions play a fundamental role in pre-
organizing the side-arms for the interaction with other species.
y Electronic supplementary information (ESI) available: molecular
modeling studies. See http://www.rsc.org/suppdata/nj/b3/b306778e/
z Dedicated to Professor Paolo Dapporto, a great teacher and friend,
on his 60th birthday.
DOI: 10.1039/b306778e
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Table 1 Coupling reagents for the activation of the carboxylic group
function of 3 to give 6
Entry
Coupling Reagent
Isolated Yield (%)
1
2
3
4
5
6
7
N-Hydroxysuccinimide/DCCI
DCCI
DMT-MM
45
< 5
< 5
< 5
10
t-BuCOCl/Et3N
EDCI/HOBT
EEDQ
51
51
2-Chloro-1-methyl pyridinium
iodide/Et3N
Chart 1 Ligand L.
8
9
p-NO2C6H4OH/DCCI
(Py)2S2/PPh3
CF3COOC6F5/Py
68
78
87
10
The guests considered in this work are NH4+, MeNH3
+
,
+
Me2NH2 and Me3NH+, which could interact with the multi-
ple hydrogen bonding sites of L. These guests were chosen con-
sidering that the recognition of ammonium and alkyl
ammonium ions is of fundamental importance and practical
interest, mainly in the biological fields.17 For example, nonac-
tin, a naturally occurring antibiotic that tightly binds NH4+, is
used in commercial ion selective electrodes for detecting the
cation in biological samples.18 The binding capabilities of L
toward such guests were investigated in aqueous solution
and by molecular modeling. The synthesis of the ligand is
reported together with its acid–base properties in aqueous
solution. The crystal structure of the monoprotonated species
HL+ and the location of the acidic protons in the several
protonated species are presented.
(2,2-dimethylpropanoyl chloride)/Et3N,23 DMT-MM (4-(4,6-
dimethoxy-1,3,5-thiazin-2-yl)-4-methylmorpholinium chloride)/
NMM (N-methylmorpholine),24 and EDCI (N-(3-dimethyl-
aminopropyl)-N0-ethylcarbodiimide)/HOBT (1-hydroxyben-
zotriazole)25 (entries 2–5), whereas the use of EEDQ
(2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline)26inCH3CN
(entry 6), or N-methyl-2-chloropyridinium iodide/Et3N27
(entry 7) gave the product 6 in approx. 50% yield. Better
results were obtained when the acid 3 was activated as
p-nitrophenyl ester28 (68%, entry 8), or when the reaction
was performed in the presence of (PyS)2(2,20-dipyridyldisul-
fide)/PPh3 (triphenilphosphine)29 (78%, entry 9); but careful
purification by column chromatography was necessary in
both cases. However, excellent results were obtained when
the acid function in 3 was activated as pentafluorophenyl
ester30 (87%, entry 10).
Results and discussion
Synthesis
Thus, treatment of 3 with pentafluorophenyl trifluoroace-
tate/Py in DMF gave, in almost quantitative yield, the corre-
sponding activated ester, which was immediately reacted with
5 with i-Pr2EtN in dry DMF at room temperature overnight.
A single crystallisation of the crude reaction mixture gave
the bis-acylated product 6 in 77% yield, which was used with-
out any further purification. Additional product (10%) was
isolated from the residue by column chromatography.
Subsequent removal of the protecting benzyl group by cata-
lytic hydrogenolysis was achieved under pressure (3 atm) and
acid conditions (Pd(OH)2/C, CH3COOH), or at atmospheric
pressure in MeOH, in the presence of 10% Pd/C cat. The sec-
ond method afforded the free ligand in almost quantitative
yield as a whitish solid, which was purified as a white dihy-
drochloride salt. The desired ligand was obtained in 61% over-
all yield from the 2,3-dihydroxypyridine, and the methodology
allows the preparation of the ligand in gram quantities.
Scheme 1 outlines the synthesis of the new multidentate ligand
L, which was based on the coupling of the protected acid
3 with the 1,7-dimethyl-1,4,7,10-tetraazacyclododecane 5,19
followed by the removal of the hydroxyl function protection.
The 3-(benzyloxy)-1-(carboxymethyl)-2(1H)-pyridinone
3
(Scheme 1) was prepared from the commercially available
2,3-dihydroxy pyridine 1 in 71% yield following the method
of Taylor et al.20,21 with minor modifications. The key cou-
pling step was studied in detail employing different coupling
reagents, and the most significant results are reported in
Table 1. Initial attempts to achieve coupling between the above
acid 3, as the corresponding succinimide derivative,20 and the
tetraamine 5 gave the bis-acylated product 6 in moderate yields
(45%, entry 1, Table 1).
Poor yields were obtained with coupling reagents
such as DCCI (N,N0-dicyclohexylcarbodiimide),22 t-BuCOCl
Scheme 1 Synthesis of L.
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Finally, following the report of Taylor et al.21 concerning
the activation of the carboxylic function in 1-carboxymethyl-
3-hydroxy-2(1H)-pyridinone as phthalimidohydroxy ester
and the coupling with N,N,N-triethylamine, we tried the same
conditions for the direct preparation of L. In our case, only a
negligible amount of the free ligand L was obtained using the
N-benzohydroxysuccinimide/DCCI system, whereas a 13%
yield was obtained when the carboxylic function was activated
with pentafluorophenyl trifluoroacetate/Py.
their mean plane (the displacement is 0.866(4) A) towards the
O(8) water molecule, with which it forms an H-bond interac-
˚
˚
tion (2.913 A). The water molecule labeled as O(11) is outside
the parallelepipedon cavity and interacts with both O(1) and
˚
O(6) (being the distances 2.909(7) and 2.906(4) A respectively):
it lies behind the plane formed by the pyridinone oxygen atoms
˚
(the displacement is 1.888(5) A), opposite to the O(10) atom.
Finally the water molecule O(9) interacts with the carbonyl
˚
oxygen atom O(3) (2.774(4) A). There are also two intermole-
cular contacts both involving the carbonyl atom O(5): it inter-
acts with O(5) and O(7) of symmetry related molecules both of
these reported by the operation ꢁx, ꢁy, ꢁz + 2. The distances
Description of the structure
˚
are respectively 3.070(6) and 2.726(5) A. The oxygen atom
[HL]Clꢂ8H2O: the asymmetric unit of [HL]Clꢂ8H2O contains a
molecule of the monocharged ligand, a chloride ion and eight
water molecules. The conformation of the [12]aneN4 ring can
be described by the sequence of its dihedral angles as [2424]
C corners,31 thus resulting that the nitrogen atoms N(3) and
N(5) are closer to each other than are the N(2) and N(4) atoms
O(4) doesn’t take part in any H-bond interaction.
Molecular modelling
The following relevant results (all the calculations were per-
formed using a distance-dependent dielectric constant set to
80) were obtained:32
˚
˚
(2.785(4) A vs. 5.427 (4) A) (Fig. 1). The former couple inter-
acts via hydrogen bonding: the acidic hydrogen atom is bound
i) The ligand, both in the neutral (L) and in the monoproto-
nated (HL+) form, appears quite rigid: both the 12-membered
ring and the side arms are characterized by stiffness.
The ligand in the neutral species prefers, quite surprisingly,33
a [2424] C corner conformation31 which is preserved in HL+.
Geometry optimizations performed on different conformations
of simplified models of L and HL+ (1,7-dimethyl-4,10-di-
acethyl-1,4,7,10-tetraazacyclododecane in the neutral and
mono-protonated forms) revealed that the stereochemistry of
the two sp2 nitrogen atoms could be the origin of the observed
rectangular shape of the uncharged ring.34
˚
to N(3) (N(3)–H(N3) bond distance 1.19(7) A) and only
˚
1.64(8) A far from N(5), the angle N(3)–H(N3)–N(5) is
158(6)ꢃ.
As expected the nitrogen atoms N(2) and N(4) have a sp2
hybridization as provided by the sum of the values of the bond
angles around them (both are ca. 360ꢃ).
The two pyridinone rings are in a plane with the amidic
=
C O bonds almost perpendicular to the mean plane and iso-
oriented. This latter plane forms an angle of 79.4(1)ꢃ with
the mean plane passing through the nitrogen atoms N(2),
N(3), N(4) and N(5). The [12]aneN4 ring and the oxygen atoms
O(2) and O(5) of the pyridinone rings, define a parallelepiped
shaped cavity within the ligand. Its dimensions could be delim-
Concerning the mobility of the side arms, inspections of MD
trajectories showed that the two pyridinone rings are virtually
facing each other throughout the simulations: the average
ꢃ
˚
˚
ited by the distances N(2)–N(4) (5.427 A) and N(3)–N(5)
angle between them being 60 , while about 6 A separate their
centroids.
˚
(2.785 A) concerning the base of the parallelepiped, while the
height of the polyhedron has been calculated as the distance
between the geometrical centroid of the base (involving the
four nitrogen atoms) and the geometrical centroid of the
ii) The energy profile concerning the reorientation of the
amide bonds of HL+ shows that the cis and the trans isomers
(cis isomer has the carbonyl groups iso-oriented while the trans
one has the CO moieties pointing in opposite directions) have
almost the same energy content, thus they both appear to be
likely in solution. The cis species having the two C!O vectors
pointing in the same direction with respect to the N!H one
(which is almost identical to the X-ray structure) is slightly
destabilized by about 1.6 kcal molꢁ1. The height of the barrier
to be overcome for the trans!cis interconversion is about 16
In summary, the dynamic behavior of HL+ (as well as L)
shows a relative 3D arrangement which produces a high
number of preorganised bonding sites meeting just outside
the cavity of the ligand, thus indicating that the ligand could
˚
oxygen atoms O(2) and O(5): its value is about 7 A. In this
region of space are located two water molecules O(10) and
O(7) (Fig. 1). The first one is located in the center of the cavity
delimited by the [12]aneN4 ring and the O(1) and O(6) oxygen
atoms: with respect to the geometrical centroid calculated with
the above mentioned nitrogen atoms, this water molecule is
˚
shifted upward, towards the pyridinone rings, of 1.37 A.
O(10) interacts with the atoms O(1) and O(6) via hydrogen
kcal molꢁ1
.
˚
bonds, being the distances 2.809(5) and 2.907(5) A, respec-
tively. Concerning the water molecule O(7), it has been found
that it is almost equidistant from the four oxygen atoms of the
˚
pyridinone rings (the mean distance is 3.055 A) and lies above
Fig. 1 Front and side view of HL+ cation with five coordinated water molecules.
New J. Chem., 2003, 27, 1575–1583
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is similar to that found for the free tetraaza-macrocyclic base
bind small charged species such as metal ions or polyatomic
cations near the molecular surface.
To verify the latter hypothesis, the complexation properties
(log K1 ¼ 10.76),17 suggesting an involvement of the amine
functions in the stabilization of the first proton. Considering
the disposition of the four protonable sites located far from
each other, the low value of the fourth protonation constant
suggests that the remaining protonable site in the HL+ species
must be strongly involved in a H-bond network, thus limiting
its availability.
of HL+ with respect to different HG+ species (i.e. HG+
¼
NH4+, MeNH3+, Me2NH2 and Me3NH+) were tested by
means of MD simulations.35
+
The results obtained are summarized below:
+
i) HL+ interacts with the complex cations NH4 and
+
MeNH3
,
whereas no stable adducts are formed with
UV-VIS absorption electronic spectra of L in aqueous solu-
tions recorded at different pH values gave further information
about the role of the pyridinone moieties in the acid–base
behavior of L. The spectra show different features in acid or
basic pH fields; at pH ¼ 2, where the H2L2+ species is pre-
valent in solution (Fig. 2), two lmax at 236 (e ¼ 9200
cmꢁ1molꢁ1dm3) and 299 nm (e ¼ 13 900 cmꢁ1molꢁ1dm3) can
be observed, while at pH higher than 7.0 two new bands with
lmax at 254 and 312 nm appear, reaching their maximum
absorbance values at pH ¼ 10 (e254 ¼ 10 500 cmꢁ1molꢁ1dm3,
e312 ¼ 16 500 cmꢁ1molꢁ1dm3); no further changes were
observed in the spectra at more strongly alkaline pH. These
different spectral features can be ascribed to the presence in
solution of the neutral form of the pyridinone moieties at
low pH values and to their deprotonated form at alkaline
pH values.15c In monitoring the spectra from acidic to basic
pH, the appearance of the new bands at 254 and 312 nm,
due to the pyridinonate moiety, was found to occur at pH over
7.0, where the L species starts to exist in solution, reaching its
maximum values in absorbance at pH ¼ 10 where the Hꢁ1Lꢁ
species has fully formed, thus proving that the deprotonation
of the pyridinones occurs in the L and in the Hꢁ1Lꢁ sꢁp2ecies.
Finally, the last deprotonation step, in which the Hꢁ2L spe-
cies is formed, must involve the amine base, taking into
account that the spectral feature does not change at pH values
above 10 where such species start to form.
Me2NH2 and Me3NH+, thus implying that at least three
intermolecular hydrogen bonds are essential to stabilize the
HL+/HG+ complex. Stable HL+/HG+ adducts show all the
six oxygen atoms of the ligand cooperating in the substrate
binding.
+
It is noteworthy that the overall shape of HL+ does not
change significantly upon complexation with HG+ and that
it closely resembles that found in the above reported MD
results32 (whose calculations were performed in mimicked aqu-
eous solution using a distance dependent dielectric constant).
This implies that the use of e ¼ 1 is, in this case, somewhat
justified.
ii) There is no doubt that the pyridinone moieties play a key
role in stabilizing the adducts, in particular the –OH functional
groupings. In fact, substitution of both the –OH binding sites
of the ligand with species unable to form H-bonds leads to the
lack of formation of any adducts. Particularly, MD simula-
tions showed that at least one hydroxyl group is essential for
the adduct stabilization. In contrast, the presence of the amide
oxygen atoms does not seem crucial in influencing the
complexation behaviour of the ligand.
Solution studies
Basicity. Table 2 summarizes the basicity constants of L as
determined potentiometrically in 0.15 mol dmꢁ3 NMe4Cl aqu-
eous solution at 298.1 K. The neutral compound L behaves
as a diprotic base and as diprotic acid under the experimental
13C and 1H NMR study of L. In order to obtain further
structural information about the protonated species, 1H and
13C NMR spectra were recorded in the pH range of the poten-
conditions used. As shown in Table 2, L can be present in solu-
2ꢁ
tiometric measurements. The 13C NMR spectrum recorded at
tion as anionic species H
L
, indicating the removal of the
ꢁ2
2ꢁ
two acidic hydrogens of the pyridinone moieties; moreover,
L can add two protons to form the H2L2+ species. By analyz-
ing the protonation constants starting from the anionic species,
it was found that L behaves as a rather strong base in the first
three protonation steps with protonation constant values ran-
ging from 10.79 for log K1 to 8.33 for log K3 , while it showed a
sharp decrease in the last step with log K4 ¼ 1.86. This trend
suggests an easy availability of protonation sites in the first
three steps in agreement with the topology of the ligands,
which is prevented in the fourth step. The value of the log K1
pH 12 and 298 K where the dianionic species H
L
ꢁ2
is preva-
lent in solution, exhibits twelve resonances in the aliphatic
range and six at higher ppm values (Fig. 3). Moreover, the
1H NMR spectrum recorded at the same temperature and
pH shows three singlets at 2.18, 2.23 and 2.28 having an inte-
gration ratio of 1 : 2 : 1, respectively, attributed to the N–CH3
resonances (see Fig. 6). The presence of three signals for the
two methyl groups of L, together with the number of aliphatic
resonances in the 13C NMR spectrum, suggests the existence of
2ꢁ
2ꢁ
two conformers in solution called Hꢁ2La
and Hꢁ2Lb
which slowly interchange on the NMR time scale. The confor-
mers are produced by the rotational restriction of the amide
Table 2 Logarithm of the protonation constants of L and formation
constants of L with the guests (HG+) NH4+, and MeNH3+ determined
by means of potentiometric measurements in 0.15 mol dmꢁ3 NMe4Cl
aqueous solutions at 298.1 K
Reaction
log K
2ꢁ
H
H
L
+ H+ ¼ Hꢁ1Lꢁ
10.79(1)a
9.08(1)
8.33(1)
1.86(2)
ꢁ2
ꢁ1Lꢁ + H+ ¼ L
L + H+ ¼ HL+
HL+ + H+ ¼ H2L2+
+
+
Reaction
NH4
CH3NH3
L + HG+ ¼ HLG+
—
2.12(3)
3.25(4)
1.69(2)
2.05(4)
2.89(3)
HL+ + HG+ ¼ H2LG2+
H2L2+ + HG+ ¼ H3LG3+
a
Values in parentheses are the standard deviations on the last signifi-
cant figure.
Fig. 2 Distribution diagram of the species of L as a function of pH,
in aqueous solution at 298.1 ꢀ 0.1 K, in 0.15 mol dmꢁ3 NMe4Cl.
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2ꢁ
Fig. 3 13C NMR spectrum of the H
L
species, recorded in aqueous solution at pH ¼ 12.
ꢁ2
groups of the tetraaza-base, as previously revealed by MM
studies. The side arms show the same frequency for both con-
formers, indicating that they are not affected by rotational
restriction. The conformer Hꢁ2La2ꢁ, showing one singlet for
the two methyl groups in both 1H and 13C NMR spectra,
and four signals for the carbon atoms of the ethylene chains,
denotes a C2 symmetry. The conformer Hꢁ2Lb2ꢁ, showing
again four signals for the carbon atoms of the ethylene chains,
but two signals for the methyl groups, both in 1H and 13C
NMR spectra, has a Cs symmetry mediated on the NMR time
scale. In substance, the two carbonyl groups can be iso-
oriented (cis) in Hꢁ2Lb2ꢁ or pointing in the opposite direction
(trans) in Hꢁ2La2ꢁ with respect to a plane passing through the
two amide nitrogen atoms of the macrocyclic base (see Fig. 4).
resonances are reported in Fig. 5a and 5b, respectively, as a
function of pH.
As shown in Fig. 5a, starting from pH 12 to pH 10 where the
species HLꢁ is prevalent, the resonances of the methyl protons
H1a, H1b, H1b0, reveal a marked downfield shift, indicating
that protonation takes place on the amine-methyl functions
The calculated relative percentage of the two conformers is
1 : 1 at 298 K for the H
2ꢁ
L
ꢁ2
species, but it changes by lower-
ing the pH. The relative percentage of the cis conformer is 59%
at pH 10, where the Hꢁ1Lꢁ species is prevalent in solution (see
Fig. 2), 62% at pH 8.4, 69% at pH 5 where the HL+ species is
prevalent, and 69% at pH 2. This means that the cis-orienta-
tion of the carbonyl groups, in agreement with the solid state
structure, is preferred with respect to the trans-one when
protonation occurs in the ligand.
The chemical shifts due to the same atoms of the different
conformers undergo approximately the same shift when the
pH is changed. This result suggests that the location of
the acidic protons in the protonated species is substantially
the same in both conformers. The trends in chemical shifts
of the positively identified and significant 1H and 13C NMR
Fig. 5 Experimental NMR chemical shifts of L in aqueous solution,
as a function of pH. 1H NMR (a); 13C NMR (b). Only the signals
clearly attributed and showing significant shifts are reported.
Fig. 4 Proposed models for the cis and trans conformers of L,
together with labels for the NMR resonances.
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The fast exchange limit for the protonated species was not
of the macrocyclic base in both conformers. This hypothesis is
in agreement with the results of the UV experiments (see
above) and with the 13C NMR resonances, which do not show
any shift for the signals due to the pyridinone groups (Fig. 5b).
Moreover, H1b0 undergoes a more marked shift than H1b,
suggesting that the nitrogen atom N10 is more involved than
N1 in the protonation of the cis-conformer. In the pH range
9–4, where the L and above all the HL+ species exists in solu-
tion, the signals of H6 and H8 undergo a downfield shift, indi-
cating an increase in the positive charge on the pyridinone
groups (Fig. 5a). These features indicate that the second and
third protonation steps, in agreement with the UV experi-
ments, involve the pyridinone groups on the oxygen atoms
bound to C9. This hypothesis is in agreement with the down-
field and upfield shifts of the C8 and C9 signals, respectively,
in the 13C NMR spectra in the same pH range (Fig. 5b).
The fourth protonation step, to obtain the H2L2+ species,
occurs below pH 3 and mainly causes a downfield shift of pro-
tons H1a and H1b in the 1H NMR spectra, suggesting that
protonation takes place on the tertiary amine groups and, in
the case of the cis-conformer, mainly involves the nitrogen
atom N1.
fully observable, denoting that the dynamic process is slightly
dependent on the protonation of the ligand. In fact, applying
the same method at pH 5.5, where the HL+ species is prevalent
in solution, we obtained approximately the same values of free
energy but different calculated activation terms: DH# ¼ 16.6
kcal molꢁ1, DS# ¼ ꢁ2.7 cal molꢁ1
K
ꢁ1, DG# ¼ 17.4 kcal
molꢁ1, respectively. The higher value of enthalpy for HL+ with
2ꢁ
respect to H
L
species could explain why free rotation was
ꢁ2
not achieved in aqueous solution for the HL+ species. These
experimental data can be ascribed to multiple factors including
the formation of several H-bond contacts in the HL+ species
also involving water molecules (see crystal structure), which
slightly increase the activation enthalpy for the free rotation
in this species.
Binding of ammonium cations. A good ligand for binding an
ammonium guest in aqueous solution must have many preor-
ganized hydrogen bonding sites. In this way, it can furnish
multiple interactions with the guest to compensate the loss
of solvation energy of ammonium. The preorganization
of side-arms of L, which produces a high number of hydrogen
bonding sites converging in a small area, as revealed by the
molecular structure and suggested by MD simulations,
induced us to explore the binding capability of the ligand
towards simple organic charged cations.
The cations studied are the series of ammonium guests
derived by ammonia, tertiary, secondary and primary methyl
amines (HG+): NH4+, MeNH3+, Me2NH2+, and Me3NH+.
The L/G systems were potentiometrically studied in 0.15
mol dmꢁ3 NMe4Cl aqueous solutions, at 298.1 K, using differ-
ent L/G molar ratios. The protonation constants of the amines
(G) were determined under our conditions and are substan-
tially in agreement with those reported in the literature.
For the G + H+ ¼ HG+ reaction, log K ¼ 9.24, 10.68, 10.81,
and 9.74 for G ¼ NH3 , MeNH2 , Me2NH, and Me3N,
respectively.
Dynamic variable-temperature NMR analysis. Variable-tem-
perature 1H NMR spectra of H
2ꢁ
L
ꢁ2
species recorded in
D2O at pH 12 in the temperature range 289–369 K show the
exchange process characteristic of the rotational restriction
of the amide groups (Fig. 6). At high temperatures, the three
sharp signals due to the N–CH3 resonances observed at room
temperature reveal a slow intermediate exchange, achieving
a
H
completely free interconversion for the equilibrium
2ꢁ
ꢁ2La ¼ Hꢁ2Lb2ꢁ up to 355 K. The rate constants at differ-
ent temperatures were calculated using the gNMR 4.0 pro-
gram36 by varying their values until satisfactory fits between
simulated and experimental data were obtained. From the
values of the rate constants at different temperatures and
applying the Eyring-plot method, a straight line was obtained
from which the activation parameters were derived.
At first glance, by examining our results for the interactions
of HG+ guests with the species of L, quantitatively reported in
Table 2 as addition constants, it is possible to observe that,
The calculated activation terms are: DH# ¼ 12.7 kcal molꢁ1
,
DS# ¼ ꢁ14.8 cal molꢁ1
K
ꢁ1, DG# ¼ 17.1 kcal molꢁ1, respec-
+
+
while NH4 and CH3NH3 form stable adducts with some
protonated species of L, no interactions were revealed for
Me2NH2+, and Me3NH+ under our experimental conditions.
These results can be explained taking into account that
dialkyl and trialkyl ammonium show lesser hydrogen bonding
links than monoalkyl-ammonium or ammonium cations; this
aspect influences not only the number of hydrogen bond
contacts but also the statistical effect in the formations of such
contacts, which is again favourable for the last two guests.
However, the thermodynamic terms related to desolvation of
host and guest and successive solvation of the adducts formed
should be taken into account.
tively. These values for the activation energies are in agreement
with those reported in the literature for the free rotation of
an amide group.37 This means that the amide groups in
2ꢁ
the H
L
ꢁ2
species are independent and, above all, that the
two side-arms do not interact with each other or with the
macrocyclic base.
NH4 and MeNH3 interact with HL+ and H2L2+ species,
while MeNH3+ interact also with the neutral species L. This is
attributed to the lower basicity of NH3 ; in fact, NH4+ coexists
in solution only with the HL+ and H2L2+ species. Both guests
show similar interaction with the same species of L however,
they have a better interaction with the more highly charged
H2L2+ species. This further aspect confirms that the binding
area of L is formed by the oxygen atoms of the preorganized
side-arms, converging in an area of small dimension located
far from the macrocyclic base in which the positive charges
are localized. In this way, the base acts as a scaffold to preor-
ganize the side-arms, allowing the interaction with these guests
in aqueous solution.
+
+
Conclusions
2ꢁ
Fig. 6 1H NMR spectra of H
L
ꢁ2
species, recorded in aqueous
The synthesis of a new ligand containing two pyridinone side-
arms attached on a tetraaza macrocyclic scaffold is reported.
solution at pH ¼ 12, recorded at different temperatures.
1580
New J. Chem., 2003, 27, 1575–1583

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Different synthetic routes for attaching side-arms were
explored and it has been found that the pentafluorophenyl
ester gave the best results.
The neutral species L behaves as diprotic base and as dipro-
tic acid in aqueous solution under the experimental conditions
used. Two conformers of L exist in solution on the NMR time
scale, showing a trans- or a cis-orientation of the two carbonyl
groups linked to the macrocyclic base. The activation para-
meters for the free interconversion of the two conformers in
under nitrogen for 48 h. Work up as described20 gave
the 1-[(ethoxycarbonyl)methyl]-3-hydroxy-2(1H)-pyridinone 2
(13.79 g, 0.07 mol) as colorless needle-like crystals; mp 150–
152 ꢃC [Lit.20 mp 150–151]; MS (EI) m/z 197 (M+, 100%),
151 (M+ ꢁ OEt, 25%), 124 (M+ ꢁ COOEt, 42%), 95 (42%),
1
68 (68%). H NMR and 13C NMR as reported.21
A solution of aq. NaOH 4M (20 mL, 0.08 mol) was added to
a solution of the above pyridinone (13.79 g, 0.07 mol) in
methanol (300 mL) and the mixture was stirred for 30 min.
After this time additional 4M NaOH was added to maintain
the pH constant at 12, followed by the addition of benzyl
chloride (28.3 mL, 0.245 mol). The mixture was refluxed for
5 h, followed by work up as reported,20 to give 3 (17.22 g,
95%; overall yield based on 2: 71%) as colorless needle-like
crystals; mp 185–186 ꢃC [Lit.20 mp 185–185.5]; MS (EI) m/z
aqueous solution were calculated. The relative ratios of the
species while, in the
2ꢁ
two conformers are 1 : 1 in the H
L
ꢁ2
protonated species, the cis-conformer prevails over the trans-,
reaching a 2 : 1 molar ratio in the HL+ species. The NMR
experiments underlined, in agreement with the UV spectra,
that the first protonation step takes place on the amine func-
tions of the macrocyclic base, while the pyridinone moieties
are involved only in the second and third steps.
1
259 (M+, 34%), 200 (M+ ꢁ CH2COOH, 49%), 91 (100%). H
NMR and 13C NMR as reported.21
Conformational studies performed by molecular modelling
showed that transformation of the two secondary amine func-
tions of the tetraaza-macrocyclic base in the amide groups stif-
4-(N), 10-(N)-bis[2-(3-benzyloxy-2-oxo-2H-pyridin-1-yl)acet-
amido]-1,7-dimethyl-1,4,7,10-tetraazacyclododecane (6). To a
well stirred solution of 3-(benzyloxy)-1-(carboxymethyl)-
2(1H)-pyridinone (2 g, 0.0077 mol) in dry DMF (5 mL) at
0 ꢃC was added pyridine (0.69 mL, 0.0085 mol) followed by
pentafluorophenyl trifluoroacetate (1.66 mL, 0.01 mol). The
reaction mixture was stirred for 1 hour at room temperature,
diluted with AcOEt (300 mL) and washed with 0.1 N aqueous
HCl (3 ꢄ 50 mL), 5% aqueous NaHCO3 (3 ꢄ 50 mL) and brine
(1 ꢄ 50 mL). The organic solution was dried over Na2SO4 and
concentrated under reduced pressure. The pentafluorophenyl
ester 4 was obtained in quantitative yield (3.28 g), and was
used without any further purification.
A solution of the above pentafluorophenyl ester 4 in dry
DMF (5 mL) was added dropwise over 30 min at 0 ꢃC to a stir-
red solution of 1,7-dimethyl-1,4,7,10-tetraazacyclododecane
5 (0.77 g, 0.00386 mol) and i-Pr2EtN (2.69 mL, 0.0155 mol) in
dry DMF (2 mL). The reaction mixture was stirred for 12 h
at room temperature. The organic yellow solution was concen-
trated under reduced pressure and the yellow oily residue was
diluted in CHCl3 and precipitated by the addition of Et2O to
give 6 (2.03 g, 77%) as a colorless solid. The organic solution
was concentrated under reduced pressure and the residue
was purified by silica gel chromatography (CH2Cl2 saturated
with NH3/methanol 99 : 1) to give additional 6 (0.26 g, 10%;
87% overall yield); mp 105–106 ꢃC dec; FTIR (film): 2957,
2924, 2850, 2792, 1652, 1601, 1455, 1252, 1064 cmꢁ1; MS
m/z (ESI): 705.3 ([M + Na]+), 683.3 ([M + 1]+); 1H NMR:
(CDCl3) d 7.49–7.25 (10H, m), 7.17–6.95 (2H, m), 6.73–6.60
(2H, m), 6.10–5.97 (2H, m), 5.08 (4H, s), 4.90–4.75 (4H, m),
3.72–3.40 (8H, m), 3.00–2.58 (8H, m), 2.55–2.19 (6H, m); 13C
NMR: (CDCl3) d 168.3, 167.4, 158.2, 148.5, 136.4, 131.0,
130.5, 128.5, 127.9, 127.4, 116.1, 115.7, 104.5, 104.4, 70.7,
58.0, 57.2, 56.4, 56.2, 50.0, 49.7, 48.2, 46.7, 43.5, 42.8, 41.7
ppm; Anal. for C38H46N6O6 (682.35): Calcd C 66.84, H 6.79,
N 12.31; Found C 66.8, H 6.8, N 12.3%.
=
fened the molecular skeleton. Moreover, the two N–C O
groups force the two 3-hydroxypyridinones to stay in the same
part of the macrocycle ring with the oxygen atoms face to
face; thus, the whole molecule appears to be preorganized
for interaction with other species.
The crystal structure reveals that the HL+ species interacts
with several water molecules via hydrogen bond contacts pro-
vided by the side-arms, suggesting that L could be a good
receptor for small substrates having high affinity towards
oxygen atoms. In fact, solution and MM studies indicate that
protonated species of L can host ammonium guests. Such
+
protonated species can discriminate between NH4 or
+
+
MeNH3 and Me2NH2 or Me3NH+, interacting only with
the first two species but not with the third and the fourth.
In conclusion, the tetraaza-macrocyclic base acts as a scaf-
fold to preorganize the side-arms, which form an electron rich
area able to stabilize guests such the ammonium cation via
hydrogen-bond interactions. Because L interacts also with
methyl ammonium or water molecules, it could also act as host
of amino acids and poly(alcohol)s, making it an attractive
ligand for molecular recognition studies.
Experimental section
General methods
UV absorption spectra were recorded at 298 K on a Varian
Cary-100 spectrophotometer equipped with a temperature
control unit. IR spectra were recorded on a Shimadzu FTIR-
8300 spectrometer. Melting points were determined on a Buchi
¨
melting point B 540 apparatus and are uncorrected. EI-MS
spectra (70 eV) were recorded with a Fisons Trio 1000 spectro-
meter; ESI mass spectra were recorded on a ThermoQuest
LCQ Duo LC/MS/MS spectrometer.
Synthesis
4-(N),10-(N)-bis[2-(3-hydroxy-2-oxo-2H-pyridin-1-yl)acet-
amido]-1,7-dimethyl-1,4,7,10-tetraazacyclododecane (L). 10%
Pd/C (0.2 g) was added to a solution of 6 (2 g, 0.0029 mol)
in dry MeOH (40 mL), and the mixture was hydrogenated at
3 atm for 15 h at room temperature. After that time, the mix-
ture was filtered over Celite and the solution was concentrated
under reduced pressure, to give L (1.43 g, 97%) as a purple
solid; mp. 120–121 ꢃC dec; MS m/z (ESI): 525.3 ([M +
Na]+), 503.3 ([M + 1]+); FTIR (film): 3509, 3406, 3005, 2959,
2924, 2850, 1652, 1583, 1552, 1497, 1467, 1274, 1239, 1006,
All chemicals were purchased from Aldrich, Fluka and Lan-
caster in the highest quality commercially available. All the
solvents were dried prior to use. Chromatographic separations
were performed on silica gel columns by flash chromatography
(Kieselgel 60, 0.040–0.063 mm, Merck). TLC analyses were
performed on precoated silica gel on aluminium sheets (Kiesel-
gel 60 F254 , Merck). Ligand L was obtained following the
synthetic procedure reported in Scheme 1. 1,4,7,10-tetraza-
cyclo-dodecane 5 was prepared as previously described.19
984 cmꢁ1 1H NMR (DMSO-d6): d 9.00 (2H, bs), 7.18–7.02
;
3-(Benzyloxy)-1-(carboxymethyl)-2(1H)-pyridinone (3). 2,3-
Dihydroxypyridine 1 (10.3 g, 0.093 mol) and ethyl bromoace-
tate (51.5 mL, 0.465 mol) were mixed together and refluxed
(2H, m), 6.83–6.68 (2H, m), 6.18–6.04 (2H, m), 5.00–4.62
(4H, m), 4.11–3.40 (8H, m), 3.40–2.70 (8H, m), 2.70–2.41
(6H, m). 13C NMR (DMSO-d6): 167.6, 167.4, 157.9, 146.6,
New J. Chem., 2003, 27, 1575–1583
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3 Crystallographic data and refinement parameters for
Table
compound L
129.7, 129.6, 115.3, 115.1, 104.8, 58.1, 57.8, 50.4, 50.3, 46.3,
46.1, 46.0, 45.8, 42.9, 42.5, 42.2 ppm.
L was dissolved in ethanol (95%) and treated with a solution
of ethanol (95%)/37% hydrochloric acid 1 : 1 to give the dihy-
drated hydrochloride salt Lꢂ2HClꢂ2H2O in almost quantitative
yield as a white solid; mp 209–210 ꢃC dec.; FTIR (film): 3502,
3396, 3002, 2959, 2921, 2850, 1650, 1581, 1551, 1465, 1408,
Empirical formula
Formula weight
Temperature (K)
[C24H35N6O6]+(Clꢁ) 8(H2O)
682.15
298
1.54184
˚
Wavelength (A)
Crystal system
Space group
triclinic
¯
P1
1272, 1134, 1074, 751 cmꢁ1
;
1H NMR: (D2O, pH ¼ 3) d
˚
a, b, c (A)
9.506(4), 9.904(3), 20.616(6)
95.45(4), 98.14(4), 118.46(3)
1659(1)
7.03 (2H, d, J ¼ 6.5 Hz), 6.95 (2H, d, J ¼ 7.2 Hz), 6.30 (2H,
dd, J ¼ 6.5, 7.2 Hz), 5.05–4.78 (4H, m), 4.03–3.52 (8H, m),
3.51–3.01 (8H, m), 3.00–2.44 (6H, m); 13C NMR: (DMSO-
d6) d 167.7, 167.6, 157.8, 146.6, 129.7, 129.6, 115.4, 115.2,
104.9, 58.4, 57.9, 57.3, 50.5, 46.0, 42.8, 42.5, 42.3; Anal. for
Lꢂ2HClꢂ2H2O, C24H40Cl2N6O8 (611.52): Calcd C 47.14, H
6.59, N 13.74; Found C 47.1, H 6.7, N 13.6.
a, b, g (ꢃ)
Volume (A )
Z, Dc(Mg mꢁ3
3
˚
)
2, 1.365
1.654
730
m(mmꢁ1
F(000)
Crystal size (mm)
)
0.4 ꢄ 0.4 ꢄ 0.6
2.20 to 60.00
5850/4826
y range (ꢃ)
Reflections collected/unique
Data/restraints/parameters
Goodness of Fit on F2
Final R indices [I > 2s(I)]
R indices (all data)
.
[HL]Cl 8H2O. Lꢂ2HClꢂ2H2O (61.1 mg, 0.1 mmol) was dis-
4082/0/416
1.045
solved in H2O (10 mL) and the pH adjusted to 6 with NaOH
0.1 M. Colorless crystals suitable for X-ray analysis formed in
a day (47 mg, 73%). Anal. for [HL]Clꢂ8H2O, C24H51ClN6O14
(647.12): Calcd C 42.20, H 7.52, N 12.30; Found C 42.1, H
7.6, N 12.2%.
R1 ¼ 0.0691, wR2 ¼ 0.1972
R1 ¼ 0.0782, wR2 ¼ 0.2090
rotating the amidic bonds j1 (C17–N2–C7–O3) and j2 (C12–
N4–C18–O4, labels refer to the X-ray structure numbering
scheme) in constant steps of 30ꢃ (range 0–360ꢃ). The resulting
144 conformations were minimized applying an external tor-
que about the dihedral angles j1 and j2. The conformers,
as well as the energy barriers associated with their mutual
interconversion, were found with the help of the UTN
program.44
MD calculations were performed at 300 K and the Verlet
leapfrog algorithm, with a time step of 1 fs, was used. Before
starting the MD procedures the initial species were optimized
until the convergence criterion was met ( < 0.01 kcal molꢁ1).
For every MD run the system was allowed to equilibrate for
50 ps and then a 500 ps dynamic study was performed (snap-
shot conformations were saved every ps). Based on the results
obtained in NMR investigations, the HL+ cis-isomer was
chosen for the HL+/HG+ studies.
X-ray crystallography
Crystallographic data for [HL]Clꢂ8H2O were collected on a
Siemens P4 diffractometer using graphite-monochromated
˚
Cu-Ka radiation (l ¼ 1.5418 A), T ¼ 298 K. Intensities data
were corrected for Lorentz and polarization effects. The struc-
ture was solved by direct methods using the SIR97 program38
and refined by full-matrix least squares against F2 using all
data (SHELX9739). An absorption correction was applied with
the program DIFABS40 once the structure was solved. Aniso-
tropic thermal parameters were used for the non H-atoms. All
the hydrogen atoms except H(O2), H(N3) and H(O5) were
introduced in calculated positions with their temperature fac-
tors refined according to the corresponding values of their
bound atoms. The atoms H(N3) and H(O5) were found in
the difference Fourier map and refined isotropically, while a
different strategy was adopted for H(O2). The latter atom
was found in the DF map, but its refinement was difficult, per-
haps because the linked oxygen atom O(2) has a quite high
temperature factor. As a consequence the position of the
H(O2) atom was refined according to the bound oxygen atom
as well as its temperature factor. The hydrogen atoms on the
water molecules could not be located in the DF map.
EMF measurements
Equilibrium constants for protonation and complexation reac-
tions with L were determined by pH-metric measurements
(pH ¼ ꢁlog [H+]) in 0.15 mol dmꢁ3 NMe4Cl at 298.1 ꢀ 0.1
K, using the fully automatic equipment that has already been
described; the EMF data were acquired with the PASAT com-
puter program.45 For the substrates investigated, their basici-
ties were determined under our experimental conditions. The
combined glass electrode was calibrated as a hydrogen concen-
tration probe by titrating known amounts of HCl with CO2-
free NMe4OH solutions and determining the equivalent point
by Gran’s method,46 which gives the standard potential Eꢃ and
the ionic product of water (pKw ¼ 13.83(1) at 298.1 K in 0.15
mol dmꢁ3 NMe4Cl, Kw ¼ [H+][OHꢁ]). At least three potentio-
metric titrations were performed for each system in the pH
range 2–11, using different molar ratio of G/L ranging from
1 : 1 to 4 : 1. All titrations were treated either as single sets or
as separate entities, for each system; no significant variations
were found in the values of the determined constants. The
HYPERQUAD computer program was used to process the
potentiometric data.47
Geometrical calculations were performed using PARST9741
and molecular plots were produced with the ORTEP3 pro-
gram.42 Table 3 reports details on the crystal data, data col-
lection, structure solution and refinement for [HL]Clꢂ8H2O.
CCDC reference number 213787. See http://www.rsc.org/
suppdata/nj/b3/b306778e/ for crystallographic data in .cif
or other electronic format.
Molecular modelling
The software used was InsightII version 98.043 provided by
MSI and implemented on an IBM RISC/6000 3AT computer.
The CVFF force field was used in all the molecular modeling
calculations.
Several modeling strategies were set up in order to enlighten
the conformational features of L and HL+.
Geometry optimizations were carried out on L and HL+ and
on several ad hoc simplified models in order to assess the role
played by the side arms and by the H-bond donor/acceptor
groupings in determining the conformational properties and
in stabilising the adducts with the complex cations (details
available as electronic supplementary informationy).
The energy profile for the amide bonds of HL+ was studied
by means of systematic conformational searches carried out by
NMR experiments
1H and 13C NMR spectra were recorded on a Bruker AC-200
instrument, operating at 200.13 and 50.33 MHz, respectively,
and equipped with a variable temperature controller. The
1582
New J. Chem., 2003, 27, 1575–1583

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Micheloni, P. Paoli, R. Pontellini and P. Rossi, Chem. Eur. J.,
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Inorg. Chem, 2001, 40, 6746; (b) J. Xu, E. Radkov, M. Ziegler
and K. N. Raymond, Inorg. Chem., 2001, 39, 4156.
14 P. W. Durbin, B. Kullgren and K. N. Raymond, Health Phys.,
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temperature of the NMR probe was calibrated using the 1,2-
ethandiol as calibration sample. For the spectra recorded in
D2O, the peak positions are reported with respect to HOD
1
(4.75 ppm) for H NMR spectra, while dioxane was used as
reference standard in 13C NMR spectra (d ¼ 67.4 ppm). For
the spectra recorded in CDCl3 and DMSO-d6 the peak posi-
tions are reported with respect to TMS. 1H–1H and 1H–13C
correlation experiments were performed to assign the signals.
Chemical shifts (d scale) are reported in parts per million
(ppm values) relative to the characteristic peak of the solvent;
coupling constants (J values) are given in hertz (Hz).
NMR spectra simulation was performed using the computer
program gNMR 4.0.36 A satisfactory fit between the simulated
and observed spectra was judged both by visual comparison
and by a least-squares fit. The activation parameters DG#,
DH#, and DS# were derived from linear regression analysis
of the Eyring plot.
18 (a) C. C andYoung, J. Chem. Educ., 1997, 74, 177; (b) J. Chin,
J. Oh, S. Y. Jon, S. H. Park, C. Walsdorff, B. Stranix, A.
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21 R. C. Fox and P. D. Taylor, Synth. Commun., 1998, 28(9), 1563.
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Acknowledgements
`
Financial support from the Italian Ministero dell’Universita e
della Ricerca (PRIN2002) and CRIST (Centro Interdipartimen-
tale di Cristallografia Strutturale, University of Florence) which
provided the X-ray equipment, are gratefully acknowledged.
24 M. Kunishima, C. Kawaki, J. Morita, K. Terao, F. Iwasaki and
S. Tani, Tetrahedron, 1999, 55, 13 159.
25 J. C. Sheehan, P. A. Cruickshank and G. L. Boshart, J. Org.
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